PROJECT SUMMARY Osteoclasts are multinucleated bone-resorbing cells and formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Osteoclasts play a crucial role in the maintenance of bone remodeling and regeneration. A group of genes regulating osteoclast differentiation have been identified, and the deregulated expression of these genes has been documented to cause various skeletal diseases. Given the fact that all genes encoding osteoclastogenic factors are expressed in the context of chromatin, a fundamental mechanism underlying osteoclast differentiation should involve chromatin regulatory pathways. Studies of transcription regulation mechanisms by chromatin reorganization may thus aid in the understanding and treatment of bone disorders caused by abnormal gene expression. MMP-9 is a member of MMP family that has been studied mainly with respect to its role in extracellular matrix remodeling. Unexpectedly, our recent studies have revealed that MMP-9 moves into the nucleus and mediates histone H3 N-terminal tail (NT) proteolysis at osteoclastogenic genes in RANKL-induced osteoclast precursor (OCP) cells. Furthermore, we found that p300/CBP-mediated H3K18 acetylation stimulates MMP-9 enzymatic activity toward H3NT in OCP- induced cells. More recent work from our laboratory also demonstrated that MMP-9 binds target nucleosomes in a manner dependent upon G9a-mediated H3K27me1 and that this binding is critical for MMP-9 recruitment and H3NT proteolysis at osteoclastogenic genes. In light of these findings, we have generated cell-permeable H3NTK27me1 mimics as a cellular tool to define H3NT residues important for MMP-9 recruitment and function. The long-term goal of the proposed research is to understand the biological processes that are controlled by H3NT proteolysis and the molecular basis of its action as an essential mediator of osteoclastogenesis. The overall objectives are to investigate MMP-9-dependent H3NT proteolysis as an osteoclastogenic signal, and to determine the molecular mechanisms whereby H3NT proteolysis activates the genes encoding master regulators of osteoclastogenesis. Our hypothesis is that MMP-9 establishes and maintains the active state of osteoclastogenic genes by a two-step mechanism wherein it gets to target genes by sensing local H3K27me1 states and cleaving H3NT in a K18ac-dependent manner. In Aim 1, we will employ the CRISPR-Cas9 system in which we can manipulate H3NT proteolysis at specific loci, and identify the genes that are directly activated by MMP-9-dependent H3NT proteolysis and necessary for proficient osteoclast differentiation. In Aim 2, we will use combined functional and structural approaches, and investigate the role of G9a-mediated H3K27me1 in the recruitment and osteoclastogenic function of MMP-9 at target genes. In Aim 3, we will examine a possible involvement of MMP-9-dependent H3NT proteolysis in disrupting nucleosome/chromatin structure, and generate a mechanistic picture for H3NT proteolysis-mediated transactivation in OCP-induced cells. In Aim 4, we will identify a group of factors that interact with H3NT and participate in pre-osteoclastogenic gene silencing, and establish MMP-9-dependent H3NT proteolysis as a key process in removing these repressors and activating transcription during osteoclastogenesis.